The indigenous multicopy miniplasmid (pXG33) of Xanthomonas campestris pv. glycines was entirely sequenced and evaluated as a cloning vector for Xanthomonas. The pXG33 contains 1738 bp and the nucleotide sequence revealed a consensus nicking site (TGATA) described for the pC194 family of rolling-circle replicating (RCR) plasmids. This nicking site is embebbed in a region of high potential to form a number of stem-loop structures. The predicted protein (Rep) showed conserved amino acid residues and potential catalytic regions, containing conserved Tyr and Glu residues. These results indicate that pXG33 replicates by a rolling-circle mechanism. For use as a cloning vector for Xanthomonas, a fragment containing the kanamycin resistance gene (aphA) and the stabilization locus (parB) was inserted into pXG33. The new construct, of 3.4 kb, was designated pXG31. By deletion of the parB locus and using pBluescript KS(+) as an intermediate, pXG40 (2.8 kb), containing unique restriction sites for BamHI, EcoRI, SacI, and KpnI at the ends of the kanamycin resistance gene, was generated. Both constructs showed stability in Xanthomonas during 18 h of growth or 72 h of fermentation, high-copy number, and no interference with pathogenicity. pXG31 and pXG40, however, were incapable of duplication in Escherichia coli and a shuttle vector (pKX33) was constructed by inactivation of some restriction sites of pXG40 and ligation to the cloning vector pBluescript KS(+). pKX33 is nonconjugative, is multicopy, is of low molecular weight (5.7 kb), presents antibiotic resistance markers for ampicillin and kanamycin, has unique restriction sites for KpnI, SalI, EcoRV, EcoRI, BamHI, XbaI, and SacI, and can be used directly for sequencing with universal primers. It can be maintained in E. coli and several species and pathovars of Xanthomonas.42126-33